The present invention relates to synthetic immunoregulatory peptides which are analogs of thymic humoral factor THF-.gamma.2 (hereinafter THF-.gamma.2) and to pharmaceutical compositions comprising them. 2. Description of the Related Art
THF-.gamma.2, an immunologically active peptide of the sequence Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu (SEQ ID No:1), has been isolated and purified from calf thymus homogenates and also prepared synthetically (U.S. Pat. No. 4,621,135; Burstein et al., 1988).
THF-.gamma.2 has been shown to possess many biological activities. For example, it was found to augment T cell functions, such as the proliferative response to the T cell mitogens phytohemagglutinin (PHA) and concanavalin A (ConA), the mixed lymphocyte reaction (MLR) and the ConA induced IL-2 production. Increased IL-2 production was shown by spleen cell cultures treated with THF-.gamma.2 prior to their triggering with ConA. THF-.gamma.2 was effective in augmenting IL-2 activity in low IL-2 producer mouse strain C3B6F1 spleen cell cultures by 100-150%, relative to control untreated cultures, at an optimal concentration of 100-300 ng/ml (Burstein et al., 1988).
THF-.gamma.2 modulates the immune state and response of human umbilical cord blood lymphocytes (UCBL). Thus, preincubation of UCBL with THF-.gamma.2 increased the percentage of cells expressing the CD4 or the CD8 differentiation antigens. THF-.gamma.2 increased likewise PHA-induced IL-2 secretion of UCBL cultures by treatment prior to suboptimal PHA stimulation. This effect was THF-.gamma.2 dose dependent with an optimum in the range of 300-600 ng/ml and was not influenced by irradiation of the UCBL (Ben-Hur et al., 1990).
THF-.gamma.2 was shown to have an effect on the immune competence of neonatally thymectomized (NTx) mice, which show a depressed immune response compared to intact age-matched mice. A biweekly course of THF-.gamma.2 injections caused a partial to complete e restoration of the immune functions as determined ex-vivo by PHA, ConA, MLR and IL-2 activity. In dose e scalation studies, using THF-.gamma.2 at doses of 4, 40 and 400 ng/Kg, the optimal daily dose was found to be 4 ng/Kg (Handzel et al., 1990). Neonatal thymectomy (NTx) of BALB/c mice caused a decrease in myeloid progenitors, which was repaired by serial injections of THF-.gamma.2. The repair of the stem cell compartment in the bone marrow correlated with an increased percentage of Thy1.sup.+ cells in the spleen of THF-.gamma.2 treated NTx mice, indicating that THF-.gamma.2 is able to regulate committed stem cell functions in the bone marrow is of immune deprived NTx mice (Pecht et al., 1993).
THF-.gamma.2 was also shown to cause restoration of immune response in mice infected with murine cytomegalovirus (MCMV), which represents a model for the stud y of the role of the immune system in the pathogenesis of human CMV. Cytomegalovirus causes T cell immune impairment in infected mice, reflected by a decreased response to the T cell mitogens PHA and ConA, reduction of ConA-induced IL-2 secretion, a marked increase in the spleen weight and development of liver focal hepatitis. Systemic treatment of MCMV-infected mice with THF-.gamma.2 resulted in a reconstitution of the mitogenic responses, IL-2 secretion, normalization of spleen weight and recovery of liver inflammation. Unlike other thymic hormones, THF-.gamma.2 did not affect interferon synthesis and NK (Natural Killer) cytotoxicity in MCMV-infected mice, suggesting that THF-.gamma.2 restores immune competence of these immunosuppressed mice through modulation of the T cell compartment (Katorza et al., 1987). Adoptive transfer experiments were performed to evaluate the prospects of enhancing the antiviral potential of MCMV immune spleen cells by THF-.gamma.2. MCMV-resistant adult BALB/c mice become highly susceptible following immunosuppression by cyclophosphamide (CY). Recipient mice were injected with MCMV and CY concomitantly and 24 hours later, adoptive transfers of syngeneic MCMV-immune spleen cells were performed. It was shown that passive transfers of MCMV immune spleen cells prevented the development of a fatal disease in 38% of the recipient mice. Daily injections of MCMV immune donor mice with THF-.gamma.2 considerably enhanced (93%) the therapeutic potential of virus specific immune cells. These results provide direct evidence for the antiviral activity of THF-.gamma.2 through its immunomodulatory effect on immune T cells. (Rager-Zisman et al., 1990)
THF-.gamma.2 did not affect IFN-.gamma. production either in vitro with cultured cells (Trainin et al., 1985) or in vivo in MCMV-infected mice (Katorza et al., 1987).
THF-.gamma.2 also has an adjuvant effect on chemotherapy in mouse plasmacytomas MOPC-315 and RPC-5. Under certain conditions, chemotherapy with antineoplastic alkylating drugs, such as cyclophosphamide or Melphalan (L-PAM), may facilitate the development of an antitumor immune response against MOPC-315 plasmacytoma. This immune promoting effect of the drug is expressed, in vivo, by cure and induction of resistance to challenge, and in vitro, by increase in specific cytotoxic potential of spleen cells from treated tumor-bearing mice. Another antineoplastic drug, 5-fluorouracil (5-FU), is capable of inducing a regression of MOPC-315 tumors, but does not lead to the development of a host antitumor response. Therefore, the influence of THF-.gamma.2 in both situations was tested, either with L-PAM or with 5-FU (Trainin et al., 1987). The effect of THF-.gamma.2 on the immune competence of 5-FU treated MOPC-315 tumor-bearing BALB/c mice was examined. Treatment of noninoculated or tumor-bearing mice with THF-.gamma.2 after 5-FU injection, resulted in an increase in the antibody response to sheep red blood cells (SRBC) and of the allogeneic response in spleen cell cultures, but had no effect on the ConA-induced IL-2 secretion over that caused by 5-FU alone. Treatment with either 5-FU alone or 5-FU and THF-.gamma.2 resulted in restoration to normal values of Lyt-1 (CD.sub.3) and L3T4 (CD.sub.4) positive populations in tumor-bearing mice. THF-.gamma.2 prolonged the survival time of MOPC-315 tumor bearing mice over that observed in mice treated with 5-FU alone (Ophir et al., 1989), and a mega dose (50 .mu.g/injection) was more effective than the low dose (0.4 ng/injection) (Ophir et al., 1991).
BALB/c mice cured from large MOPC-315 tumors by Melphalan, remain deficient in their spleen T cell function. Administration of THF-.gamma.2 to cured mice repaired their immunodeficiency as evidenced in in vitro tests with spleen cells by increase of ConA induced IL-2 secretion, allogeneic response in MLR, generation of primary antibody response and restoration of relative percentages of T cell subsets to normal values (Ophir et al., 1990).
RPC-5 murine plasmacytoma, induced by intraperitoneal injection of mineral oil, is resistant to chemotherapy with alkylating drugs. This tumor is able to induce a specific host antitumor immune response as shown by the finding that spleen cells from RPC-5 immunized mice were effective for adoptive immunotherapy (AIT) in combination with Melphalan. Treatment of RPC-5 immunized mice with THF-.gamma.2 increased the specific cytotoxic response in vitro of their spleen cells and also improved the effectiveness of these cells in AIT of RPC-5 tumor-bearing mice when performed in combination with chemotherapy by Melphalan (Ophir et al., 1990).
THF-.gamma.2 was also shown to have an effect on human granulocyte macrophage colony-forming cells (GM-CFC) and erythroid burst-forming units (BFU-E). Bone marrow (BM), peripheral blood (PB) or cord blood (CB) mononuclear cells of normal human donors were incubated overnight with various concentrations of THF-.gamma.2, washed and assayed in the GM-CFC or BFU-E agar culture assay systems stimulated by granulocyte macrophage colony-stimulation factor (GM-CSF) or erythropoietin, respectively. In several experiments lymphoid cells were removed by treatment with an anti T cell antibody, CAMPATH-1, and complement. THF-.gamma.2 significantly enhanced, in a dose-related pattern, the in vitro growth of normal human BM-GM-CFCs. This effect was GM-CSF dependent with maximal enhancement of 156% of the number of colonies relative to control cultures obtained with 25 ng/ml of THF-.gamma.2. T cell depletion by CAMPATH-1 with complement did not abrogate the THF-.gamma.2 induced enhancement of BM-GM-CFC growth. THF-.gamma.2 also exerted a significant dose response enhancement of normal PB-BFU-E growth and CB-BFU-E, an indirect effect totally abrogated by treatment with CAMPATH-1 and complement (Barak et al., 1992).
THF-.gamma.2 has a potentiating effect in vitro on myeloid progenitor cells in murine bone marrow, as determined on committed stem cells of bone marrow origin using the myeloid progenitor cell clonal assay. Preincubation of normal BM cells with THF-.gamma.2 for 1 h caused a 2-5 fold increase in the number of myeloid colonies in the presence of suboptimal concentrations of CSF but it did not replace CSF as an inducer. The optimal dose of THF-.gamma.2 causing this enhancement was in the range of 25-100 ng/ml. The superadditive effect of THF-.gamma.2 was not mediated via IL-6, since it did not induce IL-6 activity upon 24 h incubation with BM cells nor enhanced LPS-induced IL-6 secretion by bone marrow cells in vitro (Pecht et al., 1993).
THF-.gamma.2 was shown to have an effect on functions of the central nervous system. Electrophysiological experiments have demonstrated changes in the electrical activity of single neurons within the endocrine hypothalamus during an immune response. These changes can be recorded in conscious rats while the immune response is taking place. Experiments were carried out using conscious male rats bearing chronic electrodes for EEG recording and preoptic area (POA) multi-unit activity (MUA). In order to examine possible effects upon EEG and POA MUA, animals were also implanted with intracerebroventricular cannulae for administration of immune system factors. Saline (50 .mu.l) administration slightly increased POA MUA up to 45 min following injection and also increased the total time and duration of synchronized (sleep) EEG periods, while THF-.gamma.2 (0.2 ng/50 .mu.l) significantly reduced POA MUA and increased the amount and duration of synchronized EEG. These results indicate that THF-.gamma.2 is able to alter state of arousal as well as the neural activity in an area of the brain known to be of importance in the modulation of both immune and neuroendocrine activity, and that it might be useful for these purposes in clinical practice (Saphier et al., 1988).
THF-.gamma.2 was shown to be an effective immune-modulator in restoring immunodeficient aging mice. A single low dose injection of THF-.gamma.2 (16 ng/mouse) was found to enhance the frequency of ConA-responsive cells in thymus and spleen cell populations, as well as the frequency of cytokine-producing splenic T cells, up to the levels observed in young mice. The capacity of mitogen-stimulated spleen cells to produce T cell growth factor (TCGF) was also elevated. The treatment of aging mice with THF-.gamma.2 also resulted in an increased helper activity of the spleen cells to antibody producing cells (Goso et al., 1992). These results corroborate our previously reported findings on the enhancement of THF-.gamma.2 induced mitogen driven IL-2 production in mice (Katorza et al., 1987; Burstein et al., 1988) and humans (Ben-Hur et al., 1990), and restoration of helper activity to the anti-SRBC response in tumor plus chemotherapy-induced immunodeficiency (Ophir et al., 1989).
Preliminary clinical trials have been performed with synthetic THF-.gamma.2 in an open, nonrandomized pilot clinical study conducted on a group of patients with various lymphoproliferative disorders (four lymphomas, three acute lymphatic leukemias, one histiocytosis-X), one neuroblastoma, and one rhabdomyosarcoma. All were subjected to repeated pulses of combined chemotherapy and maintenance treatment, resulting in a reduction of circulating T cells and their functions. The patients were free of infection at the time of enrolment in the trial. Dosages for patients were extrapolated from the experiments on NTx mice, the lowest dosage was chosen for the clinical trials. The efficacy of treatment was evaluated by various clinical criteria, including specific signs of infection, fever rashes, general feeling of well-being, etc., and multiple laboratory parameters were monitored. Peripheral blood lymphocytes and total circulating T cells were counted. Monoclonal antibodies were used to detect T cells CD2, CD3 and their sub-populations CD4 and CD8, as well as NK cells; proliferative responses of T cells to the mitogens PHA and Con-A were compared to those of normal controls. During treatment with synthetic THF-.gamma.2, all patients, except one, were in complete clinical remission from their basic disease. No clinical benefit was expected, since patients free from complications were explicitly selected, according to the protocol of the Israel Health Ministry. Synthetic THF-.gamma.2 treatment consisted of a 3-week course of daily i.m. injections, at a dosage of 4 ng/kg/day, 6 days/wk, for a total of 18 injections. All patients were monitored for potential adverse side effects. Following treatment, T cell populations were markedly enhanced in 70% of the patients, with a trend toward normalization of CD4/CD8 ratios, and mitogenic responses were also improved. In addition, negative delayed type hypersensitivity reaction tests became positive in four of the five patients who were tested. The results demonstrated that the pattern of immune reconstitution induced by synthetic THF-.gamma.2 was similar to that obtained with the different fractions of biological THF and THF-.gamma.2. Furthermore, a marked increase of NK cell population was perceived. No side effects attributable to synthetic THF-.gamma.2 were observed during this trial.
Following the above pilot study, synthetic THF-.gamma.2 was introduced in Israel for treatment of disseminated infections, especially of viral origin. Four patients suffering from severe viral infections of the herpes group, associated with various immune impairments, were treated with synthetic THF-.gamma.2, following the same protocol described previously. A rapid regression of the viral infections was observed, similar to that seen with biological THF. A remarkable enlargement of T cell populations was observed in all cases and, as before, no deleterious side effects were documented. The results confirmed that the efficacy of synthetic THF-.gamma.2 as an immunomodulator of T cell impairments, with a marked therapeutic effect against viral infections, is identical to that seen with biological THF in prior studies (Handzel, et al., 1990).